1. Field of the Invention
The present invention relates to the field of plant biology and specifically to methods of modifying brassinosteroid levels in plants by altering levels of a specific cytochrome P450 protein involved in the brassinosteroid biosynthesis pathway. Genetic modifications of plants to increase the expression of DAS5 results in a variety of useful phenotypes such as increased fresh weight and increased overall plant size.
2. Description of the Related Art
Plant growth is coordinated by both external stimuli and internal mechanisms leading to changes in both cell division and cell expansion. In Arabidopsis, cell elongation is largely responsible for hypocotyl growth in germinating seedlings and the bolting of inflorescences at the end of vegetative growth. The main external signal is light (Deng, et al., Cell 76:423, 1994), which inhibits hypocotyl elongation and promotes cotyledon expansion and leaf development. The internal signaling mechanisms are generally regulated by plant growth substances (Klee, et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:529 1991). One class of these plant growth substances is the brassinosteroids.
Brassinosteroids produce a variety of phenotypic responses. For example, brassinosteroids accelerate seed germination and growth of seedlings, increase cell size and elongation, alter the arrangement of cortical microtubules and cellulose microfilaments, promote differentiation of xylem, promote leaf enlargement, increase plant dry weight, induce H+ export and membrane hyperpolarization, promote tissue senescence in the dark, repress anthocyanin production in light-grown plants, and induce plant pathogen resistance responses to numerous bacterial and fungal species.
Plants with altered brassinosteroid levels may be of particular economic importance to agriculture. Brassinosteroids may increase plant growth rates or alter the reproductive cycle of plants. Modification of brassinosteroid pathways may produce plants with higher crop yields and improved stress resistance (Cutler et al., Brassinosteroids: chemistry, bioactivity, and applications. ACS Symposium Series 474. Washington D.C.: American Chemical Society, 1991). Additionally, brassinosteroids may protect plants from insect attack, and qualify for classification as biochemical pesticides.
Brassinolide, one of the major brassinosteroids, was first isolated from the pollen of rape (Brassica napus) (Grove, et al., Nature, 281:216 1979), and was found to be a novel plant growth-promoting factor. To date, about 40 brassinosteroids have been found. Brassinosteroids are present at very low concentrations, and have been found to occur in all plant species examined (for review, see Mandava, et al., Ann. Rev. Plant Physiol. Plant Mol. Biol. 39:23, 1988).
Several methods for the chemical synthesis of brassinosteroids have been described. For example, U.S. Pat. No. 4,346,226 to Thompson discloses a method for producing synthetic polyhydroxylated steroidal lactones for use as plant growth promoting substances. Other methods are reviewed in Adam, et al., Phytochem. 25:1787, 1986). Historically, commercial use of the brassinosteroids for agricultural applications has been limited due to the difficulty and expense involved in producing them. In field trials, plants were found to have poor uptake of steroids through the plant epidermis, and the resulting amount of steroids required for application was considerable. Furthermore, due to the high cost of brassinolide treatments, the exogenous application of brassinosteroid compounds to increase agricultural characteristics has not been agronomically useful.
Both mutational and biochemical analyses have been beneficial in elucidating the brassinosteroid biosynthetic pathway. Several mutations have been identified that affect either light-dependent or hormone signaling pathways, resulting in plants with a dwarf phenotype. At least some of these dwarf mutants have been found to be defective in aspects of brassinosteroid biosynthesis, since they can be rescued by brassinosteroid application.
Brassinosteroids are synthesized via multiple parallel pathways (Fujioka, et al., Plant Cell Physiol. 37:1201, 1996). A multistep biosynthetic pathway leads from the sterol precursor cycloartenol to brassinolide, which is the most active of the many brassinosteroid compounds. Common plant sterols include sitosterol, stigmasterol, campesterol, 24-epicampesterol, and cholesterol are derived from cycloartenol. Most of these compounds may function as brassinosteroid precursors. A multistep pathway leads from the sterol cycloartenol to the sterol campesterol. The Arabidopsis dwf7 mutant blocks biosynthesis of 5-dehydroepisterol from episterol, while the mutant dwf5 blocks 24-methylenecholesterol biosynthesis from 5-dehydroepisterol. The mutants dim, dwf1, and 1 kb block synthesis of campesterol from 24-methylenecholesterol.
The synthesis of 5 xcex1-campestanol from campesterol is blocked in the det2 mutant of Arabidopsis. The DET2 gene encodes a steroid 5 xcex1-reductase. Overexpression of the DET2 protein increases brassinolide levels and results in larger, more robust plants. The pathway from campesterol to deoxycathasterone (or, alternatively, from 6-oxocampestanol to cathasterone) is blocked in the dwf4 mutant. The DWF4 gene and corresponding protein have been described in WO0047715 to Azpiroz. The next biosynthetic steps for each of these compounds is blocked in the mutant cpd (disclosed in U.S. Pat. No. 5,952,545 to Koncz). Both the CPD protein and DWF4 protein are types of cytochrome P450 proteins.
Once brassinolide is synthesized, it must be perceived by some cellular moiety, and then transduced to regulate developmental processes. Mutants in brassinosteroid perception are also of utility to study this aspect of brassinolide pathways. For example, three brassinosteroid insensitive mutants bri1, lka, and cu-3 accumulate brassinosteroids and impair brassinosteroid signaling. A brassinosteroid up regulated gene (BRU1) (Zurek, et al., Plant Physiol 104:505, 1994) was found to be a xyloglucan endotransglycosylase (XET), which may be involved in cell wall loosening during elongation. Brassinosteroid regulates the TCH4 gene, which is also an XET (Xu, et al., Plant Cell 7:1555, 1995).
The brassinosteroid insensitive mutant, as well as its allelic mutant cbb2 (Kauschmann, et al., Plant Jour. 9:701, 1996) were found to belong to the leucine-rich-repeat receptor-like kinase family of proteins (Li, et al., Cell 90:929, 1997). The BRI1 polypeptide is the brassinosteroid receptor (Wang et al., Nature, 410:380, 2001).
In one embodiment of the invention a method of producing a genetically modified plant having increased size as compared to a wild-type plant is produced by contacting a plant cell with at least one nucleic acid sequence encoding a DAS5 protein, wherein the nucleic acid sequence is operably associated with a promoter, to obtain a transformed plant cell, then producing a plant from the transformed plant cell, and finally selecting a plant exhibiting increased size.
Another embodiment of the present invention provides a genetically modified plant exhibiting increased size in comparison to a wild-type plant, wherein the genetically modified plant includes at least one exogenous nucleic acid sequence encoding a DAS5 polypeptide, wherein the amino acid sequence of the polypeptide is at least 80% homologous to SEQ ID NO: 1.
Yet another embodiment of the present invention provides a genetically modified seed which produces a plant exhibiting increased size in comparison to a wild-type plant, wherein the genetically modified seed includes at least one exogenous nucleic acid sequence encoding a DAS5 polypeptide with an amino acid sequence of at least 80% sequence homology to SEQ ID NO: 1.
In another embodiment of the present invention, a substantially purified DAS5 polypeptide having cytochrome P450 activity and functioning in the brassinolide biosynthetic pathway is provided.
In a further embodiment of the present invention, an antibody which binds to an isolated DAS5 polypeptide or antigenic fragments thereof is provided.
In yet another embodiment of the invention, an isolated DAS5 polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 is provided.